This invention relates to the use of azlactone-functional supports to provide cell selection.
Rapidly expanding knowledge in the areas of molecular and cellular biology, immunology, and genetics has led to the identification and characterization of a variety of highly specialized subpopulations of cells within many types of tissues. For example, the identification of rare xe2x80x9cstem cellsxe2x80x9d in such crucial tissues as the brain, islet cells of the pancreas, and liver have led to speculation that one day many diseased tissues may be treated by regeneration of healthy tissue following cell transplantation (Beardsley, Scientific American, June 1998, pp. 11-12). Such therapies have, in fact, already been achieved in areas such as cancer treatment. High doses of chemotherapy or radiation needed to destroy cancerous tissue often also destroy the patient""s bone marrow and, effectively, his entire immune system. A transplant of hematopoietic stem cells, previously isolated from either marrow or peripheral blood, can rescue the patient by reconstituting the bone marrow and cells of the immune system (Donahue, et al., Blood, 1996, 87, pp. 1644-1653). Purified stem cells or other specific cell populations are also believed to be important for developing a variety of immunotherapies (e.g., AIDS treatments) and gene therapy.
To fuel research, and indeed clinical applications, in these and related areas, increasingly effective methods of separation and purification of various cell populations are required. Over the years, a variety of methods of cell separation/purification have been utilized. These separation techniques have depended upon various biological and biochemical, physical, or immunological characteristics of the cell population to be separated (Esser, in xe2x80x9cCell Separation Methods and Applicationsxe2x80x9d, D. Recktenwald and A. Radbruch, Eds., Marcel Dekker, NY, N.Y., 1997, pp. 1-14). Immunoaffinity-based separations have shown considerable promise in terms of providing relatively pure preparations of specific cells. U.S. Pat. No. 5,035,994 (Civin) describes the use of a solid-phase linked monoclonal antibody which binds specifically to an antigen on human pluripotent lympho-hematopoietic stem cells to separate said stem cells from a suspension of marrow or blood cells. Pope, et al., (Bioconjugate Chem., 1993, 4, pp. 166-171) describe the use of bifunctional silane reagents to activate glass and cellulose solid supports. Goat anti-mouse antibodies are then covalently linked to the activated supports and the derivatized supports used to selectively deplete CD34+ or CD4+ mononuclear cells from peripheral blood samples. U.S. Pat. No. 5,215,927 (Berenson, et al.) describes the immunoselection of cells using an avidin-biotin recognition system. While these and other methods described in the art allow for selection and purification of selected cell populations, there is a continued need for new methods and materials that can provide those cell populations in improved purities and yields.
Azlactone-functional supports have been described to be quite useful for the immobilization of biologically active materials. For example, U.S. Pat. No. 5,403,902 (Heilmann, et al.) describes the preparation of particulate or beaded materials to which biomacromolecules such as proteins, antibodies, enzymes, etc. can be coupled. These materials are useful, for example, in the affinity chromatographic purification of proteins. U.S. Pat. Nos. 5,262,484, 5,292,514, 5,451,453, 5,486,358, and 5,510,421 all describe other azlactone-functional supports and materials and their uses. In none of these references has it been described or suggested that azlactone-functional supports might be useful for whole cell purification or selection.
PCT Patent Publication WO 94/00464 (Bitner et al.) describes the ability of azlactone-functional supports to separate proteinaceous materials from nonproteinaceous materials. This can be useful for separation and purification of biological materials. When the azlactone-functional support is contacted with a mixture of proteinaceous and nonproteinaceous materials, the proteinaceous materials react with and become coupled to the support, and the nonproteinaceous materials (e.g., nucleic acids) do not react with the support, but remain in solution. This publication does not describe the ability to separate whole cells from naturally-occurring biological fluids, but does disclose that the nonproteinaceous material retains biological activity for further processing after separation from the azlactone-functional support that has proteinaceous material coupled thereto.
PCT Patent Publication WO 94/22918 (Velander et al.) describes a method of derivatizing a porous support in a manner that distributes the ligand. Examples disclosed in the publication identify the covalent coupling of monoclonal antibodies and proteins for further biological separation processes.
U.S. Pat. No. 5,200,471 (Coleman et al.) describes a method of covalently coupling ligands to azlactone-functional supports, particularly with a method to increase the quality of the covalently coupled ligands, to retain high specific bound biological activity.
U.S. Pat. No. 5,561,097 (Gleason et al.) describes a method of covalently coupling small molecule ligands to azlactone-functional supports in a manner that can control the density and distribution of the ligands. This method is useful for the preparation of chromatographic supports.
A need exists for new materials and methods for the selection or purification of whole cells. It has now been found that azlactone-functional support materials, previously known to be useful for preparation of chromatographic supports for protein purification or for the preparation of covalently coupled ligands such as proteins, enzymes, and the like, can also serve as starting materials for the preparation of supports for whole cell selection and purification.
Briefly, one aspect of the invention provides a method for cell selection comprising the steps of (a) providing an azlactone-functional support, (b) derivatizing the azlactone-functional support with a substance that is biologically active towards a desired type of whole cell, wherein the substance is covalently coupled to the azlactone-functional support, (c) contacting the product of step (b) with a mixture containing the whole cells, (d) allowing the whole cells in the mixture to interact with and bind to the coupled biologically active substance, (e) removing a remainder of the mixture from the support, and (f) optionally, eluting the bound cells from the coupled biologically active substance to produce a purified collection of the whole cells.
xe2x80x9cSupportxe2x80x9d means any article that is or can be made azlactone-functional. Acceptable supports for use in the present invention can vary widely within the scope of the invention. A support can be porous or nonporous, depending on preferred final use. A support can be continuous or non-continuous depending on ultimate desired usage. A support can be made of a variety of materials, including supports made of ceramic, glassy, metallic, or polymeric materials or combinations of materials. A support can be flexible or inflexible depending on ultimate desired usage.
xe2x80x9cAzlactone-functionalxe2x80x9d means that a support has azlactone-functional groups on internal and/or external surfaces of such support. Thus, such reactive supports have an azlactone-functional group of the formula: 
wherein:
R1 and R2 independently can be an alkyl group having 1 to 14 carbon atoms, a cycloalkyl group having 3 to 14 carbon atoms, an aryl group having 5 to 12 ring atoms, an arenyl group having 6 to 26 carbon atoms and 0 to 3 S, N, and nonperoxidic O heteroatoms, or R1 and R2 taken together with the carbon to which they are joined can form a carbocyclic ring containing 4 to 12 ring atoms, and n is an integer 0 or 1.
xe2x80x9cCovalently coupledxe2x80x9d means chemically attached by means of a covalent bond.
xe2x80x9cBiologically active substancexe2x80x9d means substances which, once covalently coupled with and immobilized on the azlactone-functional support, are useful in providing selective or specific interaction with certain target whole cell populations.
xe2x80x9cWhole cellxe2x80x9d means a biologically active plant or animal cell that retains its structure intact during separation from other biological materials and is capable of remaining biologically active after use of the azlactone-functional support having a biologically active substance covalently coupled thereto to separate such plant or animal cell from other biological materials. Azlactone-functional supports useful for the preparation of cell selection supports include beaded or particulate supports such as those disclosed in U.S. Pat. No. 5,403,902, porous supports such as those disclosed in U.S. Pat. No. 5,344,701 and in PCT Patent Publication WO 93/06925, membrane supports such as those disclosed in U.S. Pat. No. 5,510,421, blends and articles prepared therefrom such as those disclosed in U.S. Pat. No. 5,408,002, substrates such as those disclosed in U.S. Pat. No. 5,292,514, and graft copolymers and articles prepared therefrom such as those disclosed in U.S. Pat. Nos. 5,013,795 and 5,262,484. All of these patents and publications are incorporated by reference herein.
The biologically active substance used to derivatize the azlactone-functional support via covalent coupling can interact directly with the specific whole cell population intended to be selected. Nonlimiting examples of such substances include an antibody (Ab) directed toward a specific cell surface marker (or antigen, Ag) expressed on the surface of the whole cell to be selected. Alternatively, the biologically active substance may interact with the selected whole cell population through a second, intermediary biologically active substance.
A feature of the present invention is the ease of preparation of supports useful for whole cell selection.
Another feature of the present invention is the versatility and variety of methods available for the preparation of azlactone-functional supports that can be modified to be useful for whole cell selection.
Another feature of the present invention is that the surface characteristics of the whole cell selection supports can be readily controlled to minimize or prevent nonspecific interactions with non-target whole cells.
An advantage of the invention is that target whole cell populations may be isolated which have higher purities than those isolated using conventional supports, such as those identified above in the Background of the Invention that utilize chemistries other than azlactone chemistry.
Another advantage of the present invention is that target whole cell populations may be isolated in higher yields than those isolated using conventional supports and in a manner that retains biologically activity.
Further features and advantages are disclosed in the following embodiments of the invention.
Azlactone-functional Supports
Azlactone-functional supports can be any compound or material containing or comprising at least one azlactone moiety that can be derivatized by covalent reaction with a biologically active substance. Such azlactone-functional supports are well known in the art. Preferably, the azlactone-functional support is a solid, insoluble material, wherein the term xe2x80x9cinsolublexe2x80x9d means does not dissolve in the medium from which the whole cell selection is to take place, comprising azlactone moieties on its surface which are readily available for reaction with the biologically active substance useful for whole cell selection.
Nonlimiting examples of azlactone-functional supports include beads, particulates, membranes, woven and nonwoven webs, and solid plastic articles comprising azlactone moieties on their surfaces. Such types of azlactone-functional supports are variously disclosed in the collection of U.S. Patents incorporated by reference above, all of which are owned by Minnesota Mining and Manufacturing Company (3M) of St. Paul, Minn., USA.
More particularly, beaded and particulate azlactone-functional supports are extensively described in U.S. Pat. No. 5,403,902 (Heilmann, et al.) incorporated by reference herein. These supports are prepared by reverse phase suspension polymerization processes and by dispersion polymerization processes from 2-alkenyl azlactone monomers and, optionally, comonomers and crosslinkers.
Porous azlactone-functional supports, such as membranes and nonwoven materials, are described in U.S. Pat. No. 5,344,701 (Gagnon, et al.) incorporated by reference herein. These supports are prepared by graft polymerization of azlactone monomers and, optionally, comonomers to the surfaces of preexisting supports using high-energy radiation. Alternatively, azlactone monomers, crosslinkers, and optionally comonomers are coated on the surfaces of the preexisting supports, then polymerized to produce the azlactone-functional supports.
Other porous azlactone-functional supports are described in PCT Patent Publication WO 93/06925 (Rasmussen, et al.) and copending, coassigned, U.S. patent appln. Ser. No. 08/776,601, incorporated herein by reference, in which azlactone-functional particles are incorporated into a continuous porous matrix such as a fibrillated polytetrafluoroethylene membrane or a nonwoven web.
Azlactone-functional membranes prepared by solvent phase inversion techniques are described in U.S. Pat. No. 5,510,421 (Dennison, et al.) incorporated by reference herein.
Thermoplastic azlactone-functional graft copolymers and copolymer blends are described in U.S. Pat. Nos. 5,013,795; 5,262,484; and 5,408,002 (all Coleman, et al.) incorporated herein by reference. These compositions are useful for the preparation of azlactone-functional molded plastic articles such as microtitration wells and plates, petri dishes, tubing, body implants, test tubes, centrifuge tubes, beakers, cuvettes, etc., which are also useful as substrates for the preparation of supports for whole cell selection in accordance with this invention.
Other azlactone-functional substrates useful in this invention are those disclosed in U.S. Pat. No. 5,292,514 (Capecchi, et al.) incorporated by reference herein.
Biologically Active Substances
Biologically active substances useful for the present invention can interact with the target whole cells either directly or indirectly, i.e. through the intermediacy of one or more secondary biologically active substances.
Biologically active substances capable of direct interaction with target whole cells include, but are not limited to, antibodies to whole cell surface antigens, lectins, and other proteins known to interact with whole cell surfaces. A wide variety of specific whole cell surface antigens have been identified, commonly referred to as CD antigens (xe2x80x9cCell Separation Methods and Applicationsxe2x80x9d, D. Recktenwald and A. Radbruch, Eds., Marcel Dekker, NY, N.Y., 1997, pp. 297-319). It is well within the capability of one skilled in the art to prepare antibodies to these antigens. In fact, many such antibodies are currently available from commercial sources such as RandD Systems, Minneapolis, Minn., and Boehringer Mannheim Corp., Indianapolis, Ind. Covalent coupling of these antibodies to azlactone-functional supports produces supports capable of direct interaction and selection of target whole cells via antibody-antigen bonding interactions.
Other biologically active substances immobilized on azlactone-functional supports can interact with target whole cells indirectly. For example, it is within the scope of the invention to use an intermediary biologically active substance which is minimally xe2x80x9cbifunctionalxe2x80x9d, i.e. it has functionality which exhibits a specific recognition or interaction with the target whole cell population but also has a second functionality which interacts and bonds to the biologically active substance which is coupled to the azlactone-functional support. An example of such an intermediary substance would be an antibody to a whole cell surface antigen conjugated to an antigen that specifically interacts with a second antibody immobilized on the support. Another example, and a preferred intermediary for use in this invention, is an anti-CD34+ antibody conjugated to biotin. Whole cell selection is accomplished via interaction of the antibody portion with the target whole cell and of biotin with avidin immobilized on the support.
Methods of Derivatizing Supports
Methods for the immobilization of biologically active substances on azlactone-functional supports are well known in the art, and are described in detail in the above mentioned patent references. Improved immobilization conditions for proteins and antibodies, specifically, are taught in U.S. Pat. No. 5,200,471 (Coleman, et al.) and in PCT Patent Publication WO 94/22918 (Velander, et al.) and companion copending, coassigned, U.S. patent appln. Ser. No. 08/296,588 (all incorporated herein by reference). Additionally, U.S. Pat. No. 5,561,097 (Gleason, et al.), incorporated by reference, describes techniques for controlling density of ligands coupled to azlactone-functional supports.
Usefulness of the Invention
The versatility and simplicity of immobilization conditions, together with the variety of techniques available for the preparation of azlactone-functional supports, allows the preparation and optimization of whole cell selection supports to a degree previously unknown. For example, once a whole cell mixture has been identified from which a target whole cell population is desired to be selected (e.g., bone marrow or peripheral blood), a base polymer support can be identified which exhibits minimal nonspecific binding of whole cells from that mixture. This ensures a high degree of purity in the selected whole cell population. Next, an azlactone moiety can be incorporated into the base polymer support by a variety of techniques disclosed in the patent references above to provide the active chemistry needed to covalently couple the biologically active substance to the support. Finally, an appropriate biologically active substance can be immobilized on the support to provide the whole cell selection support. Depending on the identity of the biologically active substance and the specific characteristics of the target whole cells, the amount of azlactone functionality and the amount of biologically active substance incorporated can be easily optimized by one skilled in the art within routine experimentation to produce the best whole cell selection. In addition, because of the unique characteristics of azlactone coupling chemistry, the surface characteristics of the final whole cell selection support can be manipulated during the immobilization process to further optimize the whole cell selection properties of said support. In many instances, whole cell selection supports can be prepared which provide selected whole cell populations with improved purities and separation yields as compared to those obtained using conventional supports employing polyacrylamide or polystyrene polymers or conventional binding chemistries such as cyanogen bromide or carbodiimide-based coupling.
Further advantages of this invention are illustrated by the following examples, but the particular materials and amounts thereof recited in these examples, as well as other conditions and details, should not be construed to unduly limit this invention.